About This Project
CRISPR Prime Editing (PE)-mediated gene integration holds promise for treating genetic diseases and enhancing crops but faces challenges in efficiency, payload capacity, and genetic baggage. This project engineers integrases to boost integration efficiency and capacity, enabling larger genetic manipulation. By designing orthogonal circuitry to autonomously exclude unwanted vector elements, we achieve clean, exclusive integration for impactful applications in medicine and agriculture.
Ask the Scientists
Join The DiscussionWhat is the context of this research?
CRISPR PE-mediated gene integration offers great potential for treating genetic diseases by inserting healthy genes at native loci, thus restoring normal function while preserving physiological states essential for long-term success. However, significant challenges remain: limited cargo capacity restricts large gene integration for complex diseases; insertion efficiency declines with large payloads; inconsistent outcomes across studies reduce reliability; vector elements add unnecessary complexity and genetic burden; and unstable nucleotide pairing reduces precision. These issues also impact agriculture, complicating the enhancement of complex crop traits. Overcoming these barriers is crucial to unlocking the full potential of precision editing across medicine and agriculture.
What is the significance of this project?
Current precision editing can only add small bits of DNA efficiently, limiting its use in research and treatment. This project changes that by engineering integrases that make DNA pairing more accurate, enabling larger and more precise gene edits. This allows us to target complex genes tied to diseases like cancer, neurodegenerative disorders, and cystic fibrosis, and to modify gene clusters for better crops. What makes this project unique is that it creates systems that act like smart filters, automatically removing unnecessary parts so only essential genetic material is added. This boosts safety in medical applications and simplifies plant engineering by cutting down prep work. Overall, this approach advances precision editing for medicine and agriculture.
What are the goals of the project?
This research advances CRISPR PE-mediated integration through two key aims and strategic outreach initiatives. Aim 1: Engineer integrases for enhanced performance—Evolve integrases with expanded catalytic potential using longer base pairs; achieve a twofold increase in efficiency (from 6.3%) and extend cargo capacity beyond 11,100 bp. Aim 2: Design intelligent circuitry—Validate orthogonal circuitry in Arabidopsis and soybean to automate vector element removal and ensure clean integration. Findings will be shared in high-impact journals, with targeted outreach to drive adoption. By enhancing integration efficiency, capacity, and purity, this project breaks through key bottlenecks in PE precision editing, setting the stage for precision medicine and agricultural innovations.
Budget
This ambitious project depends on crowdfunding to generate essential preliminary data, a foundation for securing future governmental grants. Given its substantial scale, we are strategically focused on securing an initial $49,000 for the first year to manage risk effectively. This first phase allocates $21,000 toward synthesizing integrase genes and circuitries to boost integration efficiency and $15,000 toward engineering integrases with enhanced specificity and capacity, addressing key limitations in Precision Editing. A $10,000 research stipend supports focused work to generate pivotal data, guiding the project’s trajectory for year two. Should first-year outcomes meet expectations, the second-year budget of $51,800 will cover deep sequencing and transformation costs, validating precision and reliability in plants and reinforcing future funding proposals. Together, these strategic investments provide the momentum needed to drive transformative advancements in precision editing.
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Project Timeline
The 2-year project, starting January 1, 2025, follows a structured, phase-based approach to ensure smooth progression. Each phase builds on prior outcomes, featuring reasonable time to address key challenges and implement innovations through crop testing. Regular reports to backers will ensure transparency and milestone tracking. By fostering stakeholder engagement, we will disseminate research, promote adoption, and lay the foundation for future breakthroughs.
Nov 11, 2024
Project Launched
Jan 01, 2025
Phase I: Integrase Selection • Screen integrase sequences from microorganisms, selecting ~300 candidates based on conserved recombination domains and prior research findings.
Feb 01, 2025
Phase II: Integrase Synthesis • Synthesize integrases, assemble ~300 corresponding integration circuitry and cargo donor vectors, and clone the CRISPR PE system.
Apr 01, 2025
Phase III: Integrase Testing • Evaluate integration frequency for each integrase in CRISPR PE-mediated integration using transient systems and quantitatively compare efficiency.
Jun 01, 2025
Phase IV: Engineering Integrases • Iteratively optimize the top 20 integrases under selective pressure with ~60 variant circuitry to expand base-pairing capability.
Meet the Team
Lingrui Zhang
I am a researcher with a strong dedication to Precision Editing and Synthetic Biology, bringing a multidisciplinary background in Molecular Biology, Genome Editing, and Epigenetics, complemented by expertise in molecular design. My academic journey began with a PhD in Optics in 2009. Shifting to Molecular Biology at Agriculture and Agri-Food Canada, I gained proficiency in advanced cloning techniques and exploring intricate molecular processes, such as the unfolded protein response across kingdoms. At Dr. Jian-Kang Zhu’s lab at Purdue University, I expanded my expertise to Epigenetics and Genome Editing, where I conceptualized, designed, and developed gene expression circuits refined through epigenetic regulation, CRISPR-targeted modifications, and optogenetic modulation. This interdisciplinary experience allows me to design, optimize, and implement complex molecular systems. I actively collaborate internationally and have published extensively in peer-reviewed journals, driving advancements in Precision Editing and Molecular Biology.
Lab Notes
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